Sm agar

P. violaceum QSvi11, (Pvio) (Kalla et al., 2011 (link)), gift from J. E. Strassmann (Washington University in St. Louis, USA) was routinely grown in KK2 (16 mM KH₂PO₄ and 4 mM K₂HPO₄), containing autoclaved Klebsiella aerogenes (K.aer) (final OD₆₀₀=8.5) and 10% HL5 shaken at 150 rpm. For some experiments cells were grown in association with Escherichia coli on 1/5^th SM agar (Formedium, UK). For multicellular development, cells were harvested from growth media and spread at 10⁶ cells/cm² on KK2 agar (1.5% agar in KK2), incubated at 4°C overnight and then at 22°C until the desired developmental stage had been reached.

The D. discoideum KAx3 strain was grown in association with Klebsiella aerogenes on 1/2 SM agar plates (20.9 g/L SM agar (Formedium), 11.25 g/L KH₂PO₄, 3.4 g/L K₂HPO₄, and 8.5 g/L agar) or in HL-5 liquid axenic medium (Formediun) with 150 rpm rotation at 22°C. The cells were harvested at late log stage (we call these condition of plates as “half clear”) and washed with KK2 buffer (16.5 mM KH₂PO₄, 3.9 mM K₂HPO₄ pH6.2) at least twice to eliminate bacteria or HL-5 medium completely. Washed cells of D. discoideum were plated on KK2 buffered 1.5% phytagel or gellan gum (Sigma) in 6 cm φ petri dishes (referred to as tester plates) directly or on filter paper at the density of 1 x 10⁸ cells /cm². M. incognita second-stage juveniles were prepared as described previously [11 (link)].
D. purpureum was a kind gift from Dr R. Kay (MRC Laboratory of Molecular Biology, UK) and Polysphondylium pallidum PN500 was a kind gift from Dr P. Schaap (University of Dundee, UK). D. fasciculatum S350 was provided by NBRP-nenkin (http://nenkin.nbrp.jp). These species were grown in association with Escherichia coli B/r on 2–5 LP agar plate. LP agar plate was made according to the previous report with modification [12 (link)]. In brief, we used lactose instead of glucose for LP plate.

S. maltophilia virulence was determined as previously described [31 (link)] using the social amoeba, D. discoideum. Strains of P. aeruginosa PT5 and K. pneumoniae KpGe were used as negative and positive controls, respectively, for each assay. From the overnight bacterial culture, the optical density (OD) at 600 nm was adjusted to 1.5 by dilution in LB. For co-cultures between bacteria and D. discoideum, Sm Agar (FORMEDIUM, Hustanton, United Kingdom) medium was used. One mL of each bacterial suspension was spread on Sm Agar and plates were allowed to dry for one hour to obtain a dry bacterial layer.
Meanwhile, cells of D. discoideum were washed twice in PAS buffer by centrifugation at 1000 g for 10 minutes. The amoebal suspension was adjusted to 2 x 10⁶ cells.mL^-1 and diluted in series to reach a final concentration of 7812 cells.mL^-1. Five μL of each serial dilution was spotted on the bacterial lawn. Plates were incubated at 22.5°C for five days and appearance of phagocytic plaques was checked at the end of the incubation time. This assay was performed in triplicate.
In order to interpret the results, we used the categories defined by Adamek et al. (2011) [23 (link)]: non virulent (less than 400 amoebae for lysis plaque formation), low-virulent (400–2500 amoebae for lysis plaque formation) and virulent (more than 2500 amoebae).

All compounds were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated. Axenic medium, SM agar, phosphate buffer (KK2) and blasticidin were obtained from ForMedium (Norfolk, UK). Penicillin-streptomycin was purchased from Gibco (Paisley, UK), decanoic acid was from Alfa Aesar (Massachusetts, USA) and all enzymes were purchased from Thermo Fisher Scientific (Hemel Hempstead, UK).